Use of micellar electrokinetic chromatography to measure palmitoylation of a peptide.
Palmitoylation is the thioester linkage of the fatty acid, palmitate (C16:0), to cysteine residues on a protein or peptide. This dynamic and reversible post-translational modification increases the hydrophobicity of proteins/peptides, facilitating protein-membrane interactions, protein-protein interactions and intracellular trafficking of proteins. Manipulation of palmitoylation provides a new mechanism for control over protein location and function, which may lead to better understanding of cell signaling disorders, such as cancer. Unfortunately, few methods exist to quantitatively monitor protein or peptide palmitoylation. In this study, a capillary electrophoresis-based assay was developed, using MEKC, to measure palmitoylation of a fluorescently-labeled peptide in vitro. A fluorescently-labeled peptide derived from the growth-associated protein, GAP-43, was palmitoylated in vitro using palmitoyl coenzyme A. Formation of a doubly palmitoylated GAP-peptide product was confirmed by mass spectrometry. The GAP-peptide substrate was separated from the palmitoylated peptide product in less than 7 min by MEKC. The rate of in vitro palmitoylation with respect to reaction time, GAP-peptide concentration, pH, and inhibitor concentration were also examined. This capillary electrophoresis-based assay for monitoring palmitoylation has applications in biochemical studies of acyltransferases and thioesterases as well as in the screening of acyltransferase and thioesterase inhibitors for drug development.
Borland
Laura M
LM
Department of Chemistry, University of North Carolina at Chapel Hill, Caudill and Kenan Laboratories, CB #3216, Chapel Hill, NC 27599, USA.
Allbritton
Nancy L
NL
D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D019922:GAP-43 Protein / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D054878:Lipoylation; D010168:Palmitates / Q000494:pharmacology; D010171:Palmitoyl Coenzyme A / Q000037:antagonists & inhibitors / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D005456:Fluorescent Dyes; D019922:GAP-43 Protein; D010168:Palmitates; D010446:Peptide Fragments; D010171:Palmitoyl Coenzyme A; C022776:2-bromopalmitate